There is a need in the zebrafish research community for efficient identification of mutations in known genes. Recently several groups have successfully identified mutations in genes by target selection, generation of libraries of mutation carrying zebrafish and the re-sequencing of PCR amplified exons. While this approach is effective, it is prohibitively expensive to undertake at a large scale. This proposal is designed to develop and apply a new sequencing technology, which promises higher efficiency and reduced cost in the detection of useful mutations and to target recovery of mutations in all known zebrafish transcription factors. Over the next five years, new generation high-throughput sequencing such as Solexa/Illumina machines will be used to identify mutations in 25,000 distinct 70bp target sites across the a library of 8400 individuals. We expect to recover at least 1500 new nonsense or disruptive splice-site alleles. The results of this pilot project could be applied to identify mutations in every protein-coding gene in zebrafish. We expect the cost to be between one- fifth to one-tenth the cost of conventional capillary sequencing. Every allele recovered under this project will be made freely available to the research community. PUBLIC HEALTH RELEVANCE: There are many human diseases that are caused by loss of normal gene functions. Increasingly, zebrafish are used to model human genetic diseases because zebrafish are easy and inexpensive to manipulate and maintain. Moreover, the availability of the genome sequence means that the zebrafish counterpart of the human gene can be readily identified. This project is designed to identify disruptive mutations in a large number of zebrafish genes encoding proteins that normally regulate gene expression. These mutations will provide models of human genetic disease that may be used to identify new drugs and therapies.